This invention relates to formulations containing pharmacologically active agents, solvents, carriers, and the like. More particularly, the invention relates to compounds and formulations that have anti-angiogenic effects.
The circulatory system serves an important role in the transport of nutrients, proteins, hormones, and other vital molecules that are necessary to maintain life. Blood vessels, which form an intricate network of pathways, represent an integral component of the circulatory system. In mammalian species, the internal surface of a blood vessel lumen is comprised of endothelial cells. These endothelial cells impart a smooth and low resistance quality to the lumenal surface. Critical to the free flow and transport of blood and blood constituents, the smooth and nonadhesive internal surface of the blood vessel increases the ease with which fluid flows. Without a smooth internal surface, blood vessels would become obstructed due to the formation of thrombi or other blockages at xe2x80x9cstickyxe2x80x9d locations on the internal walls. Complete or even partial blood vessel blockage would cause restriction of blood flow, thereby compromising the viability of living tissue served by the vessel. Thus, endothelial cells represent an important structural component of blood vessels and also provide blood vessels with a smooth internal surface.
The formation of blood vessels in vivo takes place in response to stimuli, which are provided in the form of specialized growth factors. These growth factors induce mitosis in cells already present in blood vessels. The new cells may replace nearby damaged cells, or the new cells may arrange themselves such that new blood vessels are formed. The process of growing blood vessels from endothelial cells is termed xe2x80x9cangiogenesis,xe2x80x9d which results in, among other characteristics, the vascularization of tissue.
Angiogenesis has become a central theme in promoting our understanding of how tissue grows. As indicated above, endothelial cell proliferation is not only desirable, but also necessary to carry out a number of physiological processes, for example the in utero formation of tissues and organs. In other contexts, however, angiogenesis may be harmful to the overall health of an organism. For example, continuous or uncontrolled angiogenesis can cause or exacerbate diseases such as rheumatoid arthritis, psoriasis, and certain retinopathies, e.g., diabetic retinopathy. Furthermore, angiogenesis makes tumor growth and metastasis possible by vascularizing the tumor, thereby supplying the tumor with blood and nutrients that are necessary to sustain the tumor""s growth, as well as providing routes by which tumor cells can migrate to distant parts of the body. Folkman (1986) Cancer Res. 46(2):467-473. Clearly then, the prevention or reduction of angiogenesis may be a desirable goal in treating some disorders and diseases. Compounds have been tested for their ability to inhibit or reduce angiogenesis. Inhibitors of vascular endothelial growth factor (VEGF), a protein that selectively induces mitosis of vascular endothelial cells, have been investigated. For example, U.S. Pat. No. 6,284,751 to Aiello et al. describes using inhibitors of the xcex2 isozyme of protein kinase C to counteract the effects of VEGF. Antibiotics such as minocycline have also been reported to inhibit angiogenesis. Some investigators have reported inhibition of tumor growth as well as reduction in the number of metastatic tumors following administration of minocycline in combination with radiation or chemotherapy. See Tamargo et al. (1991) Cancer Res. 51(2):672-675, and Teicher (1992) Cancer Res. 52(23):6702-6704. U.S. Pat. No. 5,843,925 to Backer et al. describes inhibition of angiogenesis upon administration of certain deoxytetracylines. Many of these angiogenesis-inhibiting compounds, however, have only been tested in vitro for their antiproliferative activity.
Thus, there remains a need to identify additional angiogenesis-inhibiting compounds that have demonstrated in vivo efficacy.
Brefeldin A (BFA) is a macrocyclic lactone first described by Haerri et al. See Haerri et al. (1963) Chem. Abs. 59:5726 h. Brefeldin A was also reported to have anti-viral properties, according to Tamura et al. (1968) J. Antibiotics 21:161-166. In recent years, brefeldin A has been studied extensively as a protein transport inhibitor. It is believed that brefeldin A can reversibly disrupt the Golgi apparatus, thereby affecting protein transport through the cytoplasm. Domes et al. (1989) J. Cell Biol. 109:61-72; Lippincott-Schwartz et al. (1991) J. Cell Biol. 112:567-577. It is now known that brefeldin A induces retrograde membrane transport from the Golgi apparatus to the endoplasmic reticulum (ER). Dinter et al. (1998) Histochem. Cell Biol. 109:571-590. Currently, brefeldin A is used by researchers primarily as a tool by which to interfere with the processing and sorting of finished proteins in order to more fully understand protein trafficking.
Due to its perceived lack of solubility as well as its resultant toxicity, brefeldin A has not yet been used clinically as an active agent in a pharmaceutical formulation. U.S. Pat. No. 4,608,078 to Acker et al. reported preparation of derivatives of brefeldin A with enhanced solubility, but these derivative compounds still exhibited toxicity due to inadequate solubility. In 1997, the preparation and antitumor activity of water-soluble derivatives of brefeldin A were disclosed in U.S. Pat. No. 5,696,154 to Malspeis et al. These derivatives were claimed to be suitable for intravenous delivery to animals and humans. However, these analogs have only been tested in vitro using minute quantities, which may be insufficient to produce desired therapeutic effects in vivo. U.S. Pat. No. 6,287,602 to Singh describes formulations comprising a Golgi apparatus disturbing agent as the active agent; a biocompatible carrier to increase the effective solubility of the active agent in the formulation and/or to provide a desired sustained release profile of the active agent; and a solvent. Formulations that lack a biocompatible carrier as well as those that are effective in inhibiting angiogenesis, however, are not disclosed.
Thus, there is a need to identify additional compounds as inhibitors of angiogenesis. Furthermore, there remains a need for additional formulations comprising Golgi apparatus disturbing agents for the treatment of disease. The current invention is directed to address these and other needs in the art by providing, inter alia, brefeldin A and related compounds as active agents in methods and formulations for inhibiting angiogenesis.
Accordingly, it is a primary object of the invention to provide a method for inhibiting angiogenesis in a patient by administering an effective angiogenesis-inhibiting amount of a Golgi apparatus disturbing agent to the patient.
It is yet another object of the invention to provide such a method wherein the Golgi apparatus disturbing agent is administered to a patient in need of anti-angiogenic therapy.
It is still another object of the invention to provide such a method wherein the Golgi apparatus disturbing agent is administered via injection, either systemically or locally.
It is a further object of the invention to provide a pharmaceutical formulation to treat a patient in need of anti-angiogenic therapy, wherein the formulation is comprised of a solvent, a pharmaceutically acceptable carrier, and a Golgi apparatus disturbing agent in a substantially noncytotoxic quantity effective to inhibit angiogenesis.
Additional objects, advantages, and novel features of the invention will be set forth in part in the description that follows, and in part, will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
In a first embodiment, then, a pharmaceutical formulation is provided comprising: a Golgi apparatus disturbing agent in a substantially noncytotoxic amount effective to inhibit angiogenesis in a patient in need of anti-angiogenic therapy; a solvent; and a pharmaceutically acceptable carrier. The Golgi apparatus disturbing agent may be any agent known to interfere with the functioning of the Golgi apparatus. Such Golgi apparatus disturbing agents include, without limitation, brefeldin A, nocodazole, ilimaquinone, bafilamycin, okadaic acid, retinoic acid, and combinations thereof. It is preferred, however, that the Golgi apparatus disturbing agent is brefeldin A.
The formulation must contain an amount of the Golgi apparatus disturbing agent that is effective to inhibit angiogenesis. Moreover, the amount used of the Golgi apparatus disturbing agent must not induce substantial cellular death as a result of cytotoxicity. As will be described below in further detail, substantial cellular death caused by cytotoxic quantities of Golgi apparatus disturbing agents is induced by the affected cells"" resultant inability to package and/or transport vital proteins intracellularly. In contrast, the inhibition of angiogenesis causes cell death, specifically that of tumor cells, due to a lack of vascularization.
Preferably, the pharmaceutical formulation contains an amount of the Golgi apparatus disturbing agent, e.g., brefeldin A, in a unit dosage form representing from about 0.0001 to about 200 mg/kg (milligrams drug per kilogram body weight of patient), more preferably from about 0.0001 to about 120 mg/kg, still more preferably from about 0.0001 mg to about 15 mg/kg, yet still more preferably from about 0.5 mg to about 15 mg/kg, with from about 1 mg to about 13 mg/kg being most preferred. It is noted that a substantially cytotoxic dose for one species of patient may not be substantially cytotoxic for another.
The solvent may be aqueous or nonaqueous and may also be organic or inorganic. The solvent, however, must be pharmaceutically acceptable and must solubilize at least a portion of the active agent, i.e., the Golgi apparatus disturbing agent.
As the formulations may be injected, either locally or systemically, the pharmaceutically acceptable carrier is preferably suitable for use in injection. Examples of preferred carriers include sodium chloride solutions, lactated Ringer""s solution, water, dextrose solutions, and combinations thereof. Additional components may also be included in the pharmaceutical formulation as will be described in more detail below.
In another embodiment, a method for treating a patient is provided comprising administering an angiogenesis-inhibiting amount of a Golgi apparatus disturbing agent to a patient. Although the method may be used to treat any patient who would benefit from inhibition of angiogenesis, the present method is particularly useful to treat individuals suffering from psoriasis, rheumatoid arthritis, retinopathy, and cellular proliferative diseases such as sarcomas, carcinomas, brain cancer, bladder cancer, breast cancer, colorectal cancer, head and neck cancer, liver cancer, myeloma, prostate cancer, and ovarian cancer. The method of treatment involves injecting the pharmaceutical formulation directly or nearly directly into the target site (e.g., an area exhibiting cellular proliferative disease).